l -Proline as a safer strategy to preserve ovarian function for patients facing gonadotoxic therapies

OBJECTIVE: To develop a safer strategy for preserving ovarian function in patients facing gonadotoxic therapies, this study evaluated the natural osmolytes l-proline and betaine as dimethyl sulfoxide (DMSO)-free alternatives for human ovarian tissue cryopreservation (OTC). METHOD: culture, assessment included estradiol secretion, glucose uptake, follicular viability (Calcein-AM), apoptosis (TUNEL), and markers of angiogenesis (CD31) and proliferation (Ki-67). RESULTS: l-Proline demonstrated cryoprotective efficacy comparable to DMSO in key follicular metrics: estradiol secretion and follicular viability were not significantly different between the l-proline and DMSO groups, and both were superior to betaine. Apoptosis was equally low in the l-proline and DMSO groups, and considerably lower than in the betaine group. In contrast, DMSO showed higher glucose uptake and expression of CD31 and Ki-67. CONCLUSION: l-Proline is an effective natural alternative to DMSO for OTC, achieving equivalent preservation of the follicular reserve and function with a favorable biosafety profile. These findings highlight its significant translational potential for enhancing the safety profile of clinical fertility preservation.